A cure for sickle cell disease will require that functional globin genes be transferred to a large percentage of hematopoietic stem cells. Because the majority of stem cells are normally not dividing, but undergo extensive proliferation during differentiation, this will require the stable integration of transferred genes into non-dividing cells. Retroviral vectors based on murine leukemia virus (MLV) are only able to transduce a small percentage of hematopoietic stem cells in large animal models. One reason for these disappointing results is that efficient transduction by MLV vectors requires cell division. Adeno-associated virus (AAV) vectors represent an alternative to MLV vectors, but our recent results show that efficient transduction by AAV vectors also requires cell proliferation. The objective of this project is to develop alternative vectors and techniques for the transduction of non-dividing cells. Retroviral vectors will be constructed based on visna virus and human foamy virus (HFV). Visna virus is a lentivirus capable of replicating in non-dividing cells, and preliminary experiments suggest that HFV vectors transduce non- dividing cells. The ability of these vectors to transduce and integrate in non-dividing cells will be tested using stationary fibroblast cultures. The transduction of hematopoietic stem cells will be tested using cultured human CD34+ cells and gene transfer experiments in mice, dogs and baboons. New techniques will also be developed for AAV vectors that increase the transduction of non-dividing cells by inducing DNA repair, and applied to the same experimental systems. Globin gene expression will be measured from visna, HFV and AAV vectors containing globin constructs and compared to MLV vectors, in order to determine the optimal vector system for sickle cell gene therapy.